Immunogenic influenza composition

ABSTRACT

Novel compositions useful as influenza immunogens are provided. The compositions enable a host response to immunogen sites normally not recognized by a host.

BACKGROUND

The current stable of licensed vaccines in the human and veterinaryarenas is generally successful against what are termed “Class Onepathogens.” Class One pathogens (such as measles, mumps and rubellaviruses) are those pathogens, which, in general: (1) infect or cause themost serious disease in infant, very young children, children, and youngadults; (2) carry a relatively stable microbial genome; (3) have anatural history of disease which results in spontaneous recovery; and(4) induce durable memory, associated with polyclonal and multi-epitopeantigen recognition.

In contrast, Class Two pathogens, such as, influenza virus, HIV-1,malaria parasites, Mycoplasma, such as those that cause tuberculosis,Trypanosomes, Schistosomes, Leishmania, Anaplasma, Enteroviruses,Astroviruses, Rhinoviruses, Norwalk viruses, toxigenic/pathogenic E.coli, Neisseria, Streptomyces, nontypeable Haemophilus influenzaviruses, Hepatitis C virus, cancer cells etc. are characterized by quiteopposite features. For example, Class Two pathogens: (1) tend to infectand are transmitted in a significantly extended host age range, withinfections occurring and reoccurring from childhood through thegeriatric period; (2) exhibit microbial genetic instability in definedregions of their genome (a hallmark of the successful evolution of suchpathogens); (3) in some cases, include spontaneous recovery of diseasethat frequently still leaves the host vulnerable to multiple repeatedannual infections and/or the establishment of either a chronic/active orchronic/latent infectious state; (4) induce oligoclonal, early immuneresponses that are directed to a very limited set of immunodominantepitopes which provide either narrow strain-specific protection, noprotection and/or enhanced infection; and (5) cause immune dysregulationfollowing infection or vaccination, e.g. epitope-blocking antibody,atypical primary immune response Ig subclasses, anamnesticcross-reactive recall and inappropriate TH1 and/or TH2 cytokinemetabolism.

At the immunologic level, very different etiologic agents can yielddiverse pathogenesis and disease outcome as observed, for example, withHIV-1 verses human rhinovirus. Highly successful immune system evadingstrategies, such as, Deceptive Imprinting, have evolved and are selectedand maintained across host and microbial taxa. Thus, the operationalfailures of the vertebrate immune system, for example, arising frompathogen Deceptive Imprinting, are fundamentally the same whetherinfected with HIV-1 or with the common cold virus for an average of 2-6times a year for 60 years.

Although some advances with regard to antigen delivery and expressionhave improved the immunogenicity of some Class Two microbial pathogens,current vaccine technologies have not readily translated into new,broadly effective and safe, licensed vaccines for use in humans. Thatmay be due, in large part, to a poor understanding of the fundamentallaws governing the vertebrate host defense system origin, repertoiredevelopment, maintenance, activation, senescence and co-evolution insimilar and dissimilar environments.

What is lacking currently in human influenza vaccine development is acomposition that induces immunity and protection which is less homotypicand subtype-dependent and would therefore not require the mixing andproduction of multiple subtypes in the current egg-based technologyproduction scheme year to year. A suitable new product is an influenzarecombinant HA or NA subunit vaccine that induces immune responsescapable of cross-neutralizing both intra-subtype antigenic variants andhetero-subtypes of influenza virus.

Influenza is a NIAID Category C pathogen and causes 36,000 deaths and220,000 hospitalizations in the U.S. every year. A respiratory disease,influenza spreads through droplets and/or contaminated fomites from thecough or sneeze of an infected person. Higher risk groups includechildren and the elderly, and having influenza commonly leads tosecondary complications of influenza-related pneumonias, upperrespiratory complications (otitis media in children) and other systemsdiseases (e.g. cardiovascular etc.). Influenza is the source of theworst pandemic in history; the Spanish flu of 1918 caused over 40million deaths worldwide. In the U.S., the annual direct medical costs(hospitalization, office visits, medication etc.) of influenza areestimated at $4.6 billion. Furthermore, each year, up to 111 millionworkdays are lost because of influenza with an associated cost toAmerican businesses of more than $7 billion a year in sick days and lostproductivity. Total direct and indirect costs (work days lost, schooldays lost etc.) of a severe influenza epidemic are at least $12 billionper year.

Influenza virus, and when attended by secondary bacterial infections,has long been known to be a cause of excess morbidity and mortality.Complications include pneumonia, bronchitis, congestive heart failure,myocarditis, meningitis, encephalitis and myositis. Some groups ofpeople at high risk for complications are those with chronic pulmonaryor cardiovascular disorders, residents of chronic care facilities,including nursing homes, and those persons 85 years and older.(Recommendations of the Advisory Committee on Immunization Practices(ACIP) for Prevention and Control of Influenza. MMWR, 1996, Vol 45; andThompson et al., JAMA 2003; 289:179-186). The geriatric population inthe United Stated has doubled between the years 1976 and 1999, and isexpected to rise over the next few years as the post World War II babyboomers age. People in that age bracket are 16 times more likely to dieof an influenza-associated disease than are persons aged 65 to 69.Another important contributing factor to the increase ofinfluenza-associated deaths in the 1990's is the predominance of theinfluenza A (H3N2) virus, a more virulent form of the recentlycirculating influenza viruses.

Influenza is a single-stranded ribonucleic acid (RNA) virus whichmutates rapidly to form new virulent strains. The strains are classifiedinto three groups, influenza A, B and C. The virus is further classifiedbased on two surface glycoproteins, hemagglutinin (HA) and neuraminidase(NA), into 15 HA and 9 NA subtypes. Recent whole genome analysis of thehuman influenza virus sponsored by the NIAID/NIH and collected between1996-2004 from New York State revealed that despite sharing the same HA,multiple, persistent, phylogenetically distinct lineages co-circulate inthe same population resulting in reassortment and the generation ofantigenically novel clades. While antigenic variance of HA is still thedominant selective pressure on human influenza A virus evolution, thefinding that antigenically novel clades emerge by reassortment amongpersistent viral lineages rather than via antigenic drift is of majorsignificance for the current dated annual method of influenza vaccinestrain selection and production (Holmes et al., PLoS Biol. 20053(9):e300).

At the heart of the problem in the annual global virus tracking programsand subsequent “reactionary” vaccine production that ensues, is theissue of antigenic variation. Antigenic variation is an evolvedmechanism to ensure rapid sequence variation of specific pathogengene(s) encoding homologues of an individual protein antigen, usuallyinvolving multiple, related gene copies, resulting in a change in thestructure of an antigen on the surface of the pathogen. Thus, the hostimmune system during infection or re-infection is less capable ofrecognizing the pathogen and must make new antibodies to recognize thechanged antigens before the host can continue to combat the disease. Asa result, the host cannot stay completely immune to the viral disease.That phenomenon stands as one of the more, if not, most formidableproblem challenging modern vaccine development today.

Not surprisingly, the immune response generated after infection orvaccination with all currently licensed vaccines is highly subtype andstrain specific. In practice, that means antibodies elicited duringnatural, experimental infection and vaccination are only capable ofneutralizing the homologous virus. The subtype/strain-specific humoralimmune response appears to be due to the relative immunodominance ofvarious antigenic sites found on the globular head of the hemagglutininmolecule (Wiley et al., Nature, 1981; 289:373-378). More specifically,the antibody response has been mapped to five major antigenic siteswithin the globular head of the HA. Of the five HA epitopes (A-E), twosites, A and B, are the most immunodominant and also were associatedwith the highest amount of amino acid hypervariability, due, in part, toreoccurring point mutations, deletions and occasional introduction ofN-linked glycosylation sites, known collectively as the “antigenicdrift” of the virus (Cox & Bender, Semin. Virol. 1995; 6:359-70; Buschet al., Sci. 286:1921, 1999; Plotkin & Dushoff, PNAS 100:7152, 2003; andMunoz & Deem, Vaccine 23, 1144, 2005).

Original antigenic sin, first described in 1953 by Francis (Ann. Int.Med., 1953, 399:203) is a primary immune response, that when boosted notby the homologous, but by a cross-reacting vaccine or incoming viralsubtype/strain, results in the newly formed antibodies reacting betterwith the previous antigen than with the incoming antigen.

The loss of immune specificity directed by that aleatory recall poses areal problem for the host immune system to mount equal and potenthumoral responses to the changing virus both during an infection andbetween infections. Thus, it is not surprising that natural infectionand vaccination fail to yield a more functional cross-reactive primaryand anamnestic immunity as the repertoire development against those lessimmunogenic epitopes, which may be most conserved and capable ofgenerating cross-strain immunity, are lower on the antigenic hierarchy.The immunologic phenomenon whereby immunodominant epitopes misdirect theimmune response away from more conserved and less immunogenic regions onan antigen was initially termed “clonal dominance” (Kohler et al., JAcquir. Immune Defic. Syndr. 1992; 5:1158-68), which later was renamedas “Deceptive Imprinting” (Köhler et al., Immunol. Today 1994(10):475-8).

The immunologic mechanisms for immunodominance behind deceptiveimprinting are not fully understood, and no one mechanism yet fullyexplains how or why certain epitopes have evolved to be immunoregulatoryand immunodominant. The range of immune responses observed in thephenomenon include the induction of highly strain/isolate-specificneutralizing antibody capable of inducing passive protection inexperimental animal model-viral challenge systems all the way to theinduction of a binding non-protective/non-neutralizing, blocking andeven pathogen-enhancing antibody that, in some cases, prevents the hostimmune system from recognizing nearby adjacent epitopes to interferingwith CD4 T cell help. The same decoying of the immune response throughimmunodominance resulting in a more narrowly focused set of epitopes isobserved with T cells of the host helper and cytotoxic cell-mediatedimmunity (Gzyl et al., Virology 2004; 318(2):493-506; Kiszka et al., J.Virol. 2002 76(9):4222-32; and Goulder et al., J. Virol. 2000;74(12):5679-90).

Vaccination is the best way to prevent the disease and the currenttrivalent killed virus and modified live (attenuated) influenza vaccinesare developed every year based on world-wide epidemiologicalsurveillance of active viral strains. Both vaccines contain influenza Aand influenza B subtypes. The licensed influenza vaccines consist ofinactivated whole or chemically split subunit preparations from twoinfluenza A subtypes (H1N1 and H3N2) and one influenza B subtype.Production of influenza vaccines involves the adaptation of the selectedvariants for high yield in eggs by serial passage or reassortment withother high-yield strains. Selected influenza viruses are grown inchicken eggs, and the influenza virions purified from allantoic fluid.Whole or split virus preparations are then killed by treatment with aninactivating agent, such as formalin. More than 90% of the United Statesmarket for the vaccine is served by two companies, Aventis Pasteur withmore than 50% market share and Chiron (PowderJect) (U. K.). Anintranasal vaccine, FluMist®, was approved and first sold in 2003.

Limitations of the currently available influenza vaccines include:

(1) Reduced efficacy in the elderly. Among the elderly, the rate ofprotection against illness is lower, especially for those who areinstitutionalized (Gorse et al., J. Infec. Dis. 190:11-19, 2004).Significant antibody responses to a trivalent subvirion influenzavaccine were observed in less than 30 percent of subjects ≥65 years ofage (Powers & Belshe, J. Infec. Dis. 167:584-592, 1993);

(2) Production in eggs. The current manufacturing process is dependenton chicken eggs. Influenza virus strains must replicate well in eggs anda large supply of eggs is required each year. Production is at risk eachyear because of the need to find a suitable virus combination;

(3) Inability to respond to late appearing and drift strains, such asA/Sydney/5/97 in the late nineties, or to respond to a potentialpandemic strain, such as the Hong Kong H5N1 virus that appeared in 1997;

(4) Protection with current whole or split influenza vaccines isshort-lived, and effectiveness wanes as genetic changes occur in theepidemic strains of influenza due to antigenic variation. Ideally, thevaccine strains are matched to the influenza virus strains causingdisease. Changes can occur in the hemagglutinin of egg-grown influenzavirus when compared to primary isolates from infected individuals(Oxford et al., J. Gen. Virol. 72:185-189, 1989; and Rocha et al., J.Gen. Virol. 74:2513-2518, 1993) reducing the potential effectiveness ofthe vaccine;

(5) The side effect of having the vaccine produced in eggs for thoseallergic to eggs; and

(6) The current licensed manufacturing system yields one vaccine perchicken egg infected with the influenza virus and the production time isapproximately 24 weeks.

Thus, the current licensed influenza vaccines do not: (1) induceantibodies capable of neutralizing the common annually recurringantigenic variants circulating during an epidemic, as well as thesub-type and reassortment viruses; (2) illicit a strong immune responsein the elderly; and (3) find wide applicability due to side effects, forexample, some vaccines cannot be administered to children.

SUMMARY OF THE INVENTION

The invention relates, in part, to novel influenza antigens withenhanced or novel immunogenicity. An influenza composition of interestcan serve as an improved vaccine, resulting from modifications providingthe virus or viral subunit antigen with a different array of and/ornewly recognizable epitopes.

The more efficient and rapid use of recombinant technology coupled to anovel immune refocusing technology resulting in subunit HA and/orcompositions greatly change the current practice of vaccine developmentby generating an influenza vaccine with improved cross-straineffectiveness, thereby obviating the need for the current practice ofglobal annual tracking of the virus, which will save millions ofdollars, diverted medical resources, including the time and labor of theannual scale-up for production and manufacturing in eggs, as well ashuman lives.

Additional features and advantages are described herein, and will beapparent from the following Detailed Description.

DETAILED DESCRIPTION OF THE INVENTION

Influenza is defined herein as virus that includes types A, B and C.Type A is the most virulent in humans and can result in either seasonalepidemics or occasionally and more rarely, more fatal pandemicsepisodes. The types are defined by a number of serotypes, which is areflection of the host immune response to antigens expressed on thevirus particle surface. Two structures on the virus surface that carrythe majority of epitopes correlated with vaccine protection are ahemagglutinin (HA or H) and a neuraminidase (NA or N). There are atleast 16 known H subtypes and at least 9 known N subtypes. HA mediatesvirus attachment and fusion. NA possesses sialidase activity.

“Wild type” refers to a naturally occurring organism. The term alsorelates to nucleic acids and proteins found in a naturally occurringorganism of a naturally occurring population arising from naturalprocesses, such as seen in polymorphisms arising from natural mutationand maintained by genetic drift, natural selection and so on, and doesnot include a nucleic acid or protein with a sequence obtained by, forexample, recombinant means.

“Immunogen” and “antigen” are used interchangeably herein as a moleculethat elicits a specific immune response of antibody (humoral-mediated)and/or T cell origin (cell-mediated), for example, containing anantibody that binds to that molecule or a CD4⁺ or CD8⁺ T cell thatrecognizes a virally-infected cell expressing that molecule. Thatmolecule can contain one or more sites to which a specific antibody or Tcell binds. As known in the art, such sites are known as epitopes ordeterminants. An antigen can be polypeptide, polynucleotide,polysaccharide, a lipid and so on, as well as a combination thereof,such as a glycoprotein or a lipoprotein. An immunogenic compound orproduct, or an antigenic compound or product is one which elicits aspecific immune response, which can be humoral, cellular or both.

A vaccine is an immunogen or antigen used to generate animmunoprotective response, that is, the response, such as, antibody,reduces the negative impact of the immunogen or antigen, or entityexpressing same, in a host. The dosage is derived, extrapolated and/ordetermined from preclinical and clinical studies, as known in the art.Multiple doses can be administered as known in the art, and as needed toensure a prolonged prophylactic or non-reactive state. The successfulendpoint of the utility of a vaccine for the purpose of the instantinvention is the resulting presence of an induced immune response (e.g.humoral and/or T cell-mediated) resulting, for example, in theproduction of serum antibody, or antibody made by the host in any tissueor organ, that binds the antigen or immunogen of interest. In someembodiments, the induced antibody in some way combines with a compound,molecule and the like carrying the cognate antigen or immunogen, ordirects the host to neutralize, reduce, prevent and/or eliminate apathogen from infecting and/or causing clinical disease.Immunoprotection for the purposes of the instant invention is thepresence of such anti-viral immune response (e.g. antibody and/or T cellthat binds the immunogen or infected cell) in an exposed host. That canbe determined using any known immunoassay, such as an ELISA and/orhemagglutinin inhibition assay. Alternatively, one can use a viralneutralization assay to ascertain presence of, for example, circulatingneutralizing anti-viral antibody. For the purposes of the instantinvention, observing immunoprotection in a host, that is, presence ofcirculating anti-influenza antibody, of at least seven days, at leastfourteen days, at least twenty-one days, at least thirty days or more isevidence of efficacy of a vaccine of interest. Alternatively, ingeneral, a hemagglutination inhibition (HI) titer of approximately 1:40against the homologous single strain of influenza used to make thevaccine can be an endpoint that signals a candidate vaccine is obtained.In an animal model, any delay in lethality following exposure can beevidence of protection for the purposes of the invention. Thus, in thecase of mice exposed to pathogenic strains of influenza, often the firstmice can succumb at about day 10 following exposure. Thus, if the firstday an exposed mouse succumbs is extended at least one day, at least twodays, at least three days or more is considered protection for thepurposes of the instant invention. The time of immunoprotection can beat least 14 days, at least 21 days, at least 28 days, at least 35 days,at least 45 days, at least 60 days, at least 3 months, at least 4months, at least 5 months, at least 6 months, at least 1 year, at least2 years or longer. Preferably the immunoprotection is observed inoutbred populations, and to different forms, subtypes, strains,variants, alleles and the

“Immunodominant epitope” is an epitope that selectively provokes animmune response in a host to the effective or functional exclusion,which may be partial or complete, of other epitopes on and of thatantigen.

“To immunodampen an epitope” is to modify an epitope to substantiallyprevent the immune system of the host from producing antibodies, helperor cytotoxic T cells against that epitope. However, immunodampen doesnot necessarily result in the complete removal of said epitope orreactivity to that epitope.

Immune refocusing (IR) or immune refocusing technology (IRT) can be usedto create effective vaccines against pathogens expressing Immunodominantepitopes. The technique is applied most appropriately in organisms thathave evolved a strategy known as Deceptive Imprinting to evade the hostimmune response, for example, by having an immunodominant epitope thatdisplays a high level of antigenic drift. Such an immunodominant epitopeordinarily takes the form of a plurality of amino acids that can bechanged without affecting the survivability of the pathogenic organism.

Immunodampening of an immunodominant epitope of an antigen can result inthe production in a host organism of high titer antibodies or T cellresponses against non-dominant epitopes on that antigen and/or newtiters of antibodies or T cell responses to otherwise relatively immunesilent epitopes. Such immunodampened antigens can serve as effectivevaccines against organisms that have an antigen with a moderately orhighly variable and/or conserved immunodominant epitope(s).

An immunodominant epitope can be identified by examining serum or T cellreactivity from a host organism infected with the pathogenic organism.The serum is evaluated for content of antibodies that bind to theidentified antigens that are likely to cause an immune response in ahost organism. If an immunodominant epitope is present, substantiallymany antibodies in the serum will bind to the immunodominant epitope,with little or no binding to other epitopes present on or in theantigen.

After an immunodominant epitope has been identified, the immunodominantepitope is immunodampened as taught herein using the materials andmethods taught herein and as known in the art as a design choice. Suchmanipulations can be made at the nucleic acid level, at the level of theprotein, at the level of a carbohydrate and so on, or combinationsthereof, practicing methods taught herein and known in the art.

For example, the presence of N-linked carbohydrate (CHO) can bedetermined by the primary amino acid sequence of the polypeptide. Atriplet amino acid sequence consisting of asparagine, followed by anyamino acid, and ending with a serine or threonine (N—X—S/T), where X isany amino acid other than proline or aspartic acid, is a target forN-linked CHO addition. An N-linked glycosylation site can be added orremoved from an epitope practicing methods and materials known in theart.

For example, a recombinant gp120 of HIV that displays a molecularlyintroduced N-linked sequon (NXT/S), which resulted in the addition of asupernumerary N-linked glycan in the immunodominant V3 domain, exhibitednovel antigenic properties, such as the inability to bind antibodiesthat recognize wild type V3 epitopes while inducing antibody responsesto other previously silent or less immunogenic epitopes. Presence of thesupernumerary carbohydrate moiety did not compromise the infectiousviability of the HIV-1 recombinant virus. Test animals immunized withthe recombinant glycoprotein showed moderate to high titers ofantibodies that neutralize infection to both homologous and heterologouswildtype HIV-1 in vitro. Thus, immunodampening of the immunodominantepitope within the V3 domain of gp120/160 caused the immune response torefocus on other neutralizing epitopes that are located on the sameantigen, see U.S. Pat. Nos. 5,585,250 and 5,853,724.

Alternatively, a particular amino acid of the immunodominant epitope canbe replaced, substituted or deleted to dampen immunogenicity.Immunodampening can occur by replacing, substituting or deleting oneamino acid, two amino acids, three amino acids or more of theimmunodominant epitope, for example, by site-directed mutagenesis of thenucleic acid encoding the antigen. Methods for altering nucleic acidsand/or polypeptides are provided herein, and are known in the art.

Immunodampening can be affected by any of a variety of techniques suchas, altering, substituting or deleting specific amino acids of theepitope, or adding, for example, a glycosylation site at or near theepitope. As taught herein, the changes can be effected at the level ofthe polypeptide or at the level of the polynucleotide, practicingmethods known in the art. Thus, a polypeptide can be altered by adding,deleting or substituting one or more molecules, groups, compounds andthe like to a target site on or in an epitope. For example, a particularamino acid can be derivatized chemically or can be modified to carry anextra group, such as a polysaccharide, such as, polyethylene glycol.

Following manipulation of immunogenic structures, a screening analysisof binding of the mutein to defined, known antibody that binds to one ormore immunodominant epitopes of influenza can be used to determinewhether immunodampening occurred. For example, a polypeptide can besynthesized to contain one or more changes to the primary amino acidsequence of the immunodominant epitope. Alternatively, the nucleic acidsequence of the immunodominant epitope can be modified to express animmunodampened epitope. Hence, the nucleic acid sequence can be modifiedby, for example, site-directed mutagenesis to express amino acidsubstitutions, insertions, deletions and the like, some of which mayintroduce further modification at or near the immunodominant epitope,such as, introducing a glycosylation site, such as, mutations whichcause N-glycosylation or O-glycosylation at or near the immunodominantepitope and so on.

One procedure for obtaining epitope muteins (a mutant epitope thatvaries from wild type) and the like is “alanine scanning mutagenesis”(Cunningham & Wells, Science 244:1081-1085 (1989): and Cunningham &Wells, Proc. Natl. Acad. Sci. USA 84:6434-6437 (1991)). One or moreresidues are replaced by alanine (Ala) or polyalanine residue(s). Thoseresidues demonstrating functional sensitivity to the substitutions thencan be refined by introducing further or other mutations at or for thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. Similar substitutions can be attemptedwith other amino acids, depending on the desired property of the scannedresidues.

A more systematic method for identifying amino acid residues to modifycomprises identifying residues involved in immune system stimulation orimmunodominant antibody recognition and those residues with little or noinvolvement with immune system stimulation or immunodominant antibodyrecognition. An alanine scan of the involved residues is performed, witheach Ala mutant tested for reducing immune system stimulation to animmunodominant epitope or immunodominant antibody recognition. Inanother embodiment, those residues with little or no involvement inimmune system stimulation are selected to be

Even more substantial modification in the ability to alter the immunesystem response away from the immunodominant epitope can be accomplishedby selecting an amino acid that differs more substantially in propertiesfrom that normally resident at a site. Thus, such a substitution can bemade while maintaining: (a) the structure of the polypeptide backbone inthe area of the substitution, for example, as a sheet or helicalconformation; (b) the charge or hydrophobicity of the molecule at thetarget site, or (c) the bulk of the side chain.

For example, the naturally occurring amino acids can be divided intogroups based on common side chain properties:

(1) hydrophobic: methionine (M or Met), alanine (A or Ala), valine (V orVal), leucine (L or Leu) and isoleucine (I or Ile);

(2) neutral, hydrophilic: cysteine (C or Cys), serine (S or Ser),threonine (T or Thr), asparagine (N or Asn) and glutamine (Q or Gln);

(3) acidic: aspartic acid (D or Asp) and glutamic acid (E or Glu);

(4) basic: histidine (H or His), lysine (K or Lys) and arginine (R orArg);

(5) residues that influence chain orientation: glycine (G or Gly) andproline (P or Pro), and

(6) aromatic: tryptophan (W or Trp), tyrosine (Y or Tyr) andphenylalanine (F or Phe).

Non-conservative substitutions can entail exchanging an amino acid withan amino acid from another group. Conservative substitutions can entailexchange of one amino acid for another from within a group.

Preferred amino acid substitutions are those which dampen animmunodominant epitope, but can also include those which, for example:(1) reduce susceptibility to proteolysis, (2) reduce susceptibility tooxidation, (3) alter immune system stimulating activity and/or (4)confer or modify other physico-chemical or functional properties of suchanalogs. Analogs can include various muteins of a sequence other thanthe naturally occurring peptide sequence. For example, single ormultiple amino acid substitutions (preferably conservative amino acidsubstitutions) may be made in the naturally occurring sequence. Aconservative amino acid substitution generally should not substantiallychange the structural characteristics of the parent sequence (e.g., areplacement amino acid should not tend to break a helix that occurs inthe parent sequence, or disrupt other types of secondary structure thatcharacterizes the parent sequence) unless for a change in the bulk orconformation of the R group or side chain (Proteins, Structures andMolecular Principles (Creighton, ed., W. H. Freeman and Company, NewYork (1984); Introduction to Protein Structure, Branden & Tooze, eds.,Garland Publishing, New York, N.Y. (1991)); and Thornton et al. Nature354:105 (1991)).

Ordinarily, the epitope mutant with altered biological properties willhave an amino acid sequence having at least 75% amino acid sequenceidentity or similarity with the amino acid sequence of the parentmolecule, at least 80%, at least 85%, at least 90% and often at least95% identity. Identity or similarity with respect to parent amino acidsequence is defined herein as the percentage of amino acid residues inthe candidate sequence that are identical (i.e., same residue) orsimilar (i.e., amino acid residue from the same group based on commonside-chain properties, supra) with the parent molecule residues, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity.

Covalent modifications of the molecules of interest are included withinthe scope of the invention. Such may be made by chemical synthesis or byenzymatic or chemical cleavage of the molecule, if applicable. Othertypes of covalent modifications of the molecule can be introduced intothe molecule by reacting targeted amino acid residues of the moleculewith an organic derivatizing agent that is capable of reacting withselected side chains or with the N-terminal or C-terminal residue.

Also, various organic chemistry materials and methods can be practicedto modify a component of an epitope. For example, WO05/35726 teachesvarious methods for introducing, modifying, changing, replacing and soon substituents found on biomolecules.

For example, cysteinyl residues can be reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, toyield carboxylmethyl or carboxyamidomethyl derivatives. Cysteinylresidues also can be

Histidyl residues can be derivatized by reaction withdiethylpyrocarbonate at pH 5.5-7.0. p-bromophenacyl bromide also can beused, the reaction is preferably performed in 0.1 M sodium cacodylate atpH 6.0.

Lysinyl and a amino terminal residues can be reacted with succinic orother carboxylic acid anhydrides to reverse the charge of the residues.Other suitable reagents for derivatizing α-amino-containing residuesinclude imidoesters, such as, methyl picolinimidate, pyridoxalphosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea and 2,4-pentanedione, and the amino acid can betransaminase-catalyzed with glyoxylate.

Arginyl residues can be modified by reaction with one or severalconventional reagents, such as, phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione and ninhydrin. Derivatization of arginine residuesoften requires alkaline reaction conditions. Furthermore, the reagentsmay react with lysine as well as the arginine ε amino group.

The specific modification of tyrosyl residues can be made with aromaticdiazonium compounds or tetranitromethane. For example, N-acetylimidizoleand tetranitromethane can be used to form O-acetyl tyrosyl species and3-nitro derivatives, respectively.

Carboxyl side groups (aspartyl or glutamyl) can be modified by reactionwith carbodiimides (R—N═C═C—R′), where R and R′ can be different alkylgroups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl) carbodiimide or1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues can be converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively, underneutral or basic conditions. The deamidated form of those residues fallswithin the scope of the invention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of serinyl or threonyl residues,methylation of the a amino groups of lysine, arginine and histidine(Creighton, Proteins: Structure and Molecular Properties, W.H. Freeman &Co., San Francisco, pp. 79-86 (1983)), and acetylation of the N-terminalamine and amidation of any C-terminal carboxyl group.

Another type of covalent modification involves chemically orenzymatically coupling glycosides to the molecules of interest.Depending on the coupling mode used, the sugar(s) may be attached to:(a) arginine and histidine; (b) free carboxyl groups; (c) freesulfhydryl groups, such as those of cysteine; (d) free hydroxyl groups,such as those of serine, threonine or hydroxyproline; (e) aromaticresidues such as those of phenylalanine, tyrosine or tryptophan; or (f)the amide group of glutamine. Such methods are described in WO 87/05330and in Aplin & Wriston, CRC Crit. Rev. Biochem., pp. 259-306 (1981).Sugar residues also can be added enzymatically using, for example, aglucosyl transferase, a sialyl transferase, a galactosyl transferase andso on.

Removal of any carbohydrate moieties present on the molecule of interestmay be accomplished chemically or enzymatically. Chemicaldeglycosylation, for example, can require exposure of the molecule tothe compound, trifluoromethanesulfonic acid, or an equivalent compound,resulting in cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving theremainder of the molecule intact. Chemical deglycosylation is described,for example, in Hakimuddin et al., Arch. Biochem. Biophys. 259:52 (1987)and in Edge et al., Anal. Biochem. 118:131 (1981). Enzymatic cleavage ofcarbohydrate moieties on molecules can be achieved by any of a varietyof endoglycosidases and exoglycosidases as described, for example, inThotakura et al., Meth. Enzymol. 138:350(1987). Thus, a mannosidase, afucosidase, glucosaminosidase, a galactosidase and so on can be used.

RNA or DNA encoding the HA, NA and the like of influenza is readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to therelevant genes, Innis et al. in PCR Protocols. A Guide to Methods andApplications, Academic (1990), and Sanger et al., Proc. Natl. Acad. Sci.74:5463 (1977)). Once isolated, the DNA may be placed into expressionvectors, which are then placed into host cells, such as, E. coli cells,NS0 cells, COS cells, Chinese hamster ovary (CHO) cells or myelomacells, to obtain synthesis of the protein of interest in the recombinanthost cells. The RNA or DNA also may be modified, for example, bysubstituting bases to optimize for codon usage in a particular host orby covalently joining to the coding sequence of a heterologouspolypeptide.

The phrases and terms, as well as combinations thereof, “functionalfragment, portion, variant, derivative or analog” and the like, as wellas forms thereof, of an influenza virus, antigen, component, subunit,HA, NA and the like thereof relate to an element having qualitativebiological activity in common with the wild type or parental elementfrom which the variant, derivative, analog and the like was derived. Forexample, a functional portion, fragment or analog of HA is one whichstimulates an immune response as does native HA, although the responsemay be to a different epitope on the HA.

Thus, included within the scope of the invention are functionalequivalents of a virus, or portion or derivative thereof, of interest.The term “functional equivalents” includes the virus and portionsthereof with the ability to stimulate an immune response to influenza.

Parts of an influenza virus of interest, such as membrane ornon-membrane preparations carrying HA, NA, M2, or combinations, as wellas preparations of any other influenza antigens, can be obtainedpracticing methods known in the art. When one or more immunodominantnon-protective epitopes (IDNPEs, which also include epitopes thatstimulate strain-specific, but less broad immunity) are removed ordampened, for example, by intramolecular modifications (e.g. deletions,charge changes, adding one or more N-linked sequons and so on) and givenas an antigen to a naive animal, the changes to the IDNPE can induce anew hierarchy of immune responses at either or both the B and T celllevels (Garrity et al., J. Immunol. (1997) 159(1):279-89) againstsubdominant or previously silent epitopes. That technology as describedherein is known as “Immune Refocusing.”

Once a change is made, whether the change alters, such as, reduces thereactivity of the immunodominant epitope now modified, the “dampenedepitope, antigen and so on” is determined as taught herein or as knownin the art. That can be tested in vitro by determining the reactivity ofthe dampened antigen with defined antisera known to react with thatdominant epitope, such as by an ELISA or Western blot, for example.Candidates demonstrating reduced reactivity with those defined antiseraare chosen for testing in vivo to determine whether those dampenedantigens are immunogenic and the host generates an immune responsethereto. Hence, for example, a mouse is immunized to the dampenedantigen as known in the art, serum obtained and tested in an in vitroassay for reactivity therewith. That antiserum then can be tested onwild type virus to determine if the antibody still recognizes the wildtype epitope or the wild type antigen. That can be done, for example, inan ELISA or a Western blot. The latter can be informative, revealingwhether the particular immunodominant epitope is bound, and if theantiserum remains reactive with influenza, the size and possibly, theidentity of the molecule carrying the epitope reactive with the mouseantiserum.

Those candidate immunodampened antigens less or no longer reactive withknown antisera that bind to the parent immunodominant antigen, yetremain immunogenic in hosts are selected as candidate vaccines forfurther testing. Candidates may also stimulate enhanced reactivity tothe parental immunodominant antigen, while targeting immune refocusedepitopes for immune recognition. For example, the mouse antiserumthereto can be tested for reactivity with a number of influenza strainsin standardized anti-viral-based assays to determine how generic thatantibody is, that is, whether the newly recognized epitopes on thedampened antigen are generic to a wide range of influenza strains and ifthe antibody has broad antiviral activity.

Thus, a recombinant HA (rHA) subunit protein vaccine can be sufficientto protect against challenge from homologous strains of influenza virus.An rHA also can be used as an immunogen in older adults. A secondgeneration, immune refocused HA subunit vaccine as taught herein couldinduce protective immunity against heterologous strains as well (Treanoret al., J. Infectious Diseases 2006; 193:1223-8).

In one embodiment, the HA and NA of influenza were selected as targetsfor refocusing the host immune response to other non-dominant sites onHA and NA as novel targets for an immunoprotective response, preferablyone of broad scope and spectrum active on a wide variety of strains andso on.

For example, HA has five immunodominant sites or epitopes, known as A-E.Site A includes amino acids 140-146 of HA type strains and has thesequence, KRRSNKS (SEQ ID NO: 1). In the Wyoming strain, that sitealready has three glycosylation sites associated therewith as comparedto the Hong Kong strain. Thus, one approach is to remove the loopstructure defined by site A, for example, by replacement of the KRRSNKS(SEQ ID NO: 1) sequence by, for example, GG.

Site B includes amino acids 189-197 of HA with the sequence SDQISLYAQ(SEQ ID NO:2). That forms a helix which interacts with amino acids158-161 having the sequence, KYKY (SEQ ID NO:3). A number of possiblechanges can be made to the B site, such as substitute NAS for QIS;substitute NIT for SLY; substitute NST for KYK at 158; and substituteNTS for YKY at 159, all of those changes introducing an N-glycosylationsequence at those four sites.

Site C includes amino acids 276-278 having the sequence KCN. NCT cansubstitute for KCN.

Site D includes a large antiparallel loop at amino acids 201-220. Theentire loop can be deleted. Also, the glycosylation site, NIT, cansubstitute for RIT at sites 201-203.

Site E includes amino acids 79-82, FQNK (SEQ ID NO:4). The glycosylationsite, NET, can substitute for QNK.

The above changes can be combined, such as, either of the NST and NTSchanges at site B can be combined with the suggested, exemplary changesto sites C and/or E.

The above alterations to immunodominant sites can be obtained bycloning, site-directed mutagenesis, amplification and so on as known inthe art.

Thus, the A site change above can be obtained using the primers, ATop:GGAACAAGCTCTGCTTGCggcggtTTCTTTAGTAGATTGAATTGG (SEQ ID NO:5) and ABottom:CCAATTCAATCTACTAAAGAAGAAaccgcgcGCAAGCAGAGCTTGTTCC (SEQ ID NO:6) toobtain the sequence, GTSSACGGFFSRLN (SEQ ID NO:7) containing thedeletion described above and insertion of the GG dipeptide at thatdeletion site.

The B site changes can be obtained by using primers, B1Top:CAAATCAGCCTATATGCTaatGCATCAGGAAGAATCAC (SEQ ID NO:8) and B1bottom:GTGATTCTTCCTGATGCattAGCATATAGGCTGATTTG (SEQ ID NO:9) to yield thesequence QISLYANASGRI (SEQ ID NO:10); the primers B2Top:CACCACCCGGTTACGGACaatGACacAATCAGCCTATATGCTCAAGC (SEQ ID NO: 11) andB2bottom GCTTGAGCATATAGGCTGATgtGTCattGTCCGTAACCGCGTGGTG (SEQ ID NO: 12)to yield the sequence HHPVTDNDTISLYAQ (SEQ ID NO: 13): the primersB3Top: CGGACAGTGACCAAATCAatCTAtcTGCTCAAGCATCAGGAAG (SEQ ID NO: 14) andB3Bottom: CTTCCTGATGCTTGAGCAgaTAGatTGATTTGGTCACTGTCCG (SEQ ID NO: 15) toyield the sequence DSDQINLSAQASG (SEQ ID NO: 16); the primers B4top:GAATTGGTTGACCCACTTAAAtTATAcATACCCAGCATGAACGTGAC (SEQ ID NO: 17) andB4bottom: GTCACGTTCAATGCTGGGTATgTGTAaTTTAAGTGGGTCAACCAATTC (SEQ ID NO:18) to yield the sequence NWLTHLNYTYPALNV (SEQ ID NO: 19); and theprimers B5top: GAATTGGTTGACCCACTTAAAAaACAAAacCCCAGCATTGAACGTGACTAT G(SEQ ID NO:20) and B5bottom:CATAGTCACGTTCAATCTGGGgtTTTGTtTTTTAAGTGGGTCAACCAATC (SEQ ID NO:21) toyield the sequence NWLTHLKNKTPALNVTM (SEQ ID NO:22).

The C site change can be obtained using the primers C1top: AGTC4GATGCACCCATTGGCAAtTGCAgTTCTGAATGCATCACTCC (SEQ ID NO:23) andC1bottom: GGAGTGATGCATTCAGAAcTGCAaTTGCCAATGGGTGCATCTGATC (SEQ ID NO:24)to yield the sequence SDAPIGNCSSECIT (SEQ ID NO:25).

The D site change can be obtained using the primers D1Top:CTATATGCTCAAGCATCAGGAAatATCACAGTCTCTACCAAAAG (SEQ ID NO:26) and D1Bottom: CTTTTGGTAGAGACTGTGATatTTCCTGATGCTTGAGCATATAG (SEQ ID NO:27) toobtain the sequence LYAQASGNITVSTKRS (SEQ ID NO:28).

The E site change can be obtained using the primers E1Top:GATGGCTTCCAAAATAAGAcATGGGACCITITTGTTGAAC (SEQ ID NO:29) and E1bottom:GTTCAACAAAAAGGTCCCATgTCTTATTTTGGAAGCCATC (SEQ ID NO:30) to yield thesequence DGFQNKTWDLFVE (SEQ ID NO:31).

The HAS1: CAGTCCTCATCAGATCCTTG (SEQ ID NO:32), HAS2:GGTAAGGGATATCTCCAGCAG (SEQ ID NO:33) primers can be used for sequencing,with HAS3: cgcgattgcgccaaatatgcc (SEQ ID NO:34) as a negative.

Many of the antigenic sites are rich in charged amino acid residues.Another approach is to replace those charged residues by substitutingalanine residues therefor. Examples of such changes include KRR to AGAin site A; KYKY (SEQ ID NO:3) to AYKY (SEQ ID NO:35) and SDQI to SAQI(SEQ ID NO:36) in site B; KCN to ACN in site C and RIT to AIT in site D.

In addition, a mutation to assess the function of the hydrophobictyrosine residue in site B can be obtained replacing SLY with SLT.

In addition to B cell epitopes, T cell epitopes also can beimmunodampened. A major CD4 epitope in the region of residues 177 to 199comprises an MHC Class 11 binding epitope outside of the alreadytargeted B site. Mutations in the residues LYIWGVHHP (SEQ ID NO:37) todampen the T cell response include replacing LYIW with VYIW (SEQ IDNO:38) or VTIW (SEQ ID NO:39); and replacing VHHP with IHAG (SEQ IDNO:40).

To obtain approval from regulatory agencies, such as the U.S. Food andDrug Administration or European Medicines Agency for human products,biological pharmaceutics must meet purity, safety and potency standardsdefined by the pertinent regulatory agency. To produce a vaccine thatmeets those standards, the recombinant organisms can be maintained inculture medium that is, for example, certified free of transmissiblespongiform encephalopathies (herein referred to as “TSE”).

For example, plasmids harboring the vaccine-encoding sequence carry anon-antibiotic selection marker, since it is not always ideal to useantibiotic resistance markers for selection and maintenance of plasmidsin bacteria that are designed for use in humans, although a preferredembodiment relates to use of a recombinant subunit vaccine. In oneembodiment, therefore, the present invention provides a selectionstrategy in which, for example, a catabolic enzyme is utilized as aselection marker by enabling the growth of bacteria in medium containinga substrate of said catabolic enzyme as a carbon source. An example ofsuch a catabolic enzyme includes, but is not restricted to, lacYZencoding lactose uptake and β-galactosidase (Genbank Nos. J01636,J01637, K01483 or K01793). Other selection markers that provide ametabolic advantage in defined media include, but are not restricted to,galTK (GenBank No. X02306) for galactose utilization, sacPA (GenBank No.J03006) for sucrose utilization, trePAR (GenBank No. Z54245) fortrehalose utilization, xylAB (GenBank No. CAB13644 and AAB41094) forxylose utilization etc. Alternatively, the selection can involve the useof antisense mRNA to inhibit a toxic allele, such as the sacB allele(GenBank No. NP_391325).

For testing, the immunogen of interest is administered to a nonhumanmammal for the purpose of obtaining preclinical data, for example.Exemplary nonhuman mammals include nonhuman primates, dogs, cats,rodents and other mammals. Such mammals may be established animal modelsfor a disease to be treated with the formulation, or may be used tostudy toxicity of the immunogen of interest. In each of thoseembodiments, dose escalation studies may be performed in the mammal.

The specific method used to formulate the novel vaccines andformulations described herein is not critical to the present inventionand can be selected from or can include a physiological buffer (Feigneret al., U.S. Pat. No. 5,589,466 (1996)); aluminum phosphate or aluminumhydroxyphosphate (e.g. Ulmer et al., Vaccine, 18:18 (2000)),monophosphoryl-lipid A (also referred to as MPL or MPLA; Schneerson etal. J. Immunol., 147:2136-2140 (1991); e.g. Sasaki et al. Inf. Immunol.,65:3520-3528 (1997); and Lodmell et al. Vaccine, 18:1059-1066 (2000)),QS-21 saponin (e.g. Sasaki et al., J. Virol., 72:4931 (1998));dexamethasone (e.g., Malone et al., J. Biol. Chem. 269:29903 (1994));CpG DNA sequences (Davis et al., J. Immunol., 15:870 (1998));interferon-α (Mohanty et al., J. Chemother. 14(2):194-197, (2002)),lipopolysaccharide (LPS) antagonist (Hone et al., J. Human Virol., 1:251-256 (1998)) and so on.

The formulation herein also may contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely impact each other.For example, it may be desirable to further provide an adjuvant. Suchmolecules suitably are present in combination in amounts that areeffective for the purpose intended. The adjuvant can be administeredsequentially, before or after antigen administration.

The immunogen of interest can be used with a second component, such as atherapeutic moiety conjugated to or mixed with same, administered as aconjugate, separately in combination, mixed prior to use and so on as atherapeutic, see, for example, Levine et al., eds., New GenerationVaccines. 2^(nd) Marcel Dekker, Inc., New York, N.Y., 1997). Thetherapeutic agent can be any drug, vaccine and the like used for anintended purpose. Thus, the therapeutic agent can be a biological, asmall molecule and so on. The immunogen of interest can be administeredconcurrently or sequentially with a second influenza immunogeniccomposition, immunodampened or not, for example. Thus, an immunodampenedantigen of interest can be combined with an existing vaccine, althoughthat approach would minimize the use thereof if the existing vaccine ismade in eggs.

The term “small molecule” and analogous terms include, but are notlimited to, peptides, peptidomimetics, amino acids, amino acidanalogues, polynucleotides, polynucleotide analogues, carbohydrates,lipids, nucleotides, nucleotide analogues, organic or inorganiccompounds (i.e., including heterorganic and/organometallic compounds)having a molecular weight less than about 10,000 grams per mole, organicor inorganic compounds having a molecular weight less than about 5.000grams per mole, organic or inorganic compounds having a molecular weightless than about 1,000 grams per mole, organic or inorganic compoundshaving a molecular weight less than about 500 grams per mole, and salts,esters, combinations thereof and other pharmaceutically acceptable formsof such compounds which stimulate an immune response or are immunogenic,or have a desired pharmacologic activity.

Thus, the immunogen of the invention may be administered alone or incombination with other types of treatments, including a second immunogenor a treatment for the disease being treated. The second component canbe an immunostimulant.

In addition, the immunogen of the instant invention may be conjugated tovarious effector molecules such as heterologous polypeptides, drugs,radionucleotides and so on, see, e.g., WO 92/08495, WO 91/14438; WO89/12624; U.S. Pat. No. 5,314,995; and EPO 396,387. An immunogen may beconjugated to a therapeutic moiety such as an antibiotic (e.g., atherapeutic agent or a radioactive metal ion (e.g., α emitters such as,for example, ²¹³Bi)) or an adjuvant.

Therapeutic compounds of the invention alleviate at least one symptomassociated with influenza. The products of the invention may be providedin pharmaceutically acceptable compositions as known in the art or asdescribed herein. The terms “physiologically acceptable,”“pharmacologically acceptable” and so on mean approved by a regulatoryagency of the Federal or a state government or listed in the U.S.Pharmacopeia or other generally recognized pharmacopeia for use inhumans.

The products of interest can be administered to a mammal in anyacceptable manner. Methods of introduction include, but are not limitedto, parenteral, subcutaneous, intraperitoneal, intrapulmonary,intranasal, epidural, inhalation and oral routes, and if desired forimmunosuppressive treatment, intralesional administration. Parenteralinfusions include intramuscular, intradermal, intravenous, intraarterialor intraperitoneal administration. The products or compositions may beadministered by any convenient route, for example, by infusion or bolusinjection, by absorption through epithelial or mucocutaneous linings(e.g., oral mucosa, rectal and intestinal mucosa etc.) and may beadministered together with other biologically active agents.Administration can be systemic or local. In addition, it may bedesirable to introduce the therapeutic products or compositions of theinvention into the central nervous system by any suitable route,including intraventricular and intrathecal injection; intraventricularinjection may be facilitated by an intraventricular catheter, forexample, attached to a reservoir, such as an Ommaya reservoir. Inaddition, the product can be suitably administered by pulse infusion,particularly with declining doses of the products of interest.Preferably the dosing is given by injection, preferably intravenous orsubcutaneous injections, depending, in part, on whether theadministration is brief or chronic.

Various other delivery systems are known and can be used to administer aproduct of the present invention, including, e.g., encapsulation inliposomes, microparticles or microcapsules (see Langer, Science 249:1527(1990); Liposomes in the Therapy of Infectious Disease and Cancer,Lopez-Berestein et al., eds., (1989)).

The active ingredients may be entrapped in a microcapsule prepared, forexample, by coascervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsule andpoly-(methylmethacylate) microcapsule, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nanoparticles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences,16th edition, A. Osal, Ed. (1980).

Pulmonary administration can also be employed, e.g., by use of aninhaler or nebulizer, and formulation with an aerosolizing agent. Thecomposition of interest may also be administered into the lungs of apatient in the form of a dry powder composition, see e.g., U.S. Pat. No.6,514,496.

It may be desirable to administer the therapeutic products orcompositions of the invention locally to the area in need of treatment;that may be achieved by, for example, and not by way of limitation,local infusion, topical application, by injection, by means of acatheter, by means of a suppository or by means of an implant, saidimplant being of a porous, non-porous or gelatinous material, includinghydrogels or membranes, such as sialastic membranes or fibers.Preferably, when administering a product of the invention, care is takento use materials to which the protein does not absorb or adsorb.

In yet another embodiment, the product can be delivered in a controlledrelease system. In one embodiment, a pump may be used (see Langer,Science 249:1527 (1990); Sefton, CRC Crit. Ref. Biomed. Eng. 14:201(1987); Buchwald et al., Surgery 88:507 (1980); and Saudek et al., NEJM321:574 (1989)). In another embodiment, polymeric materials can be used(see Medical Applications of Controlled Release, Langer et al., eds.,CRC Press (1974); Controlled Drug Bioavailability, Drug Product Designand Performance, Smolen et al., eds., Wiley (1984); Ranger et al., J.Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983); see also Levy et al.,Science 228:190 (1985); During et al., Ann. Neurol. 25:351 (1989); andHoward et al., J. Neurosurg. 71:105 (1989)). In yet another embodiment,a controlled release system can be placed in proximity of thetherapeutic target.

Sustained release preparations may be prepared for use with the productsof interest. Suitable examples of sustained release preparations includesemi-permeable matrices of solid hydrophobic polymers containing theimmunogen, which matrices are in the form of shaped articles, e.g.,films or matrices. Suitable examples of such sustained release matricesinclude polyesters, hydrogels (for example,poly(2-hydroxyethylmethacrylate), poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid andethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers (such as injectable microspherescomposed of lactic acid-glycolic acid copolymer) andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release cells, proteins and productsfor and during shorter time periods. Rational strategies can be devisedfor stabilization depending on the mechanism involved.

The compositions can take the form of solutions, suspensions, emulsion,tablets, pills, capsules, powders, sustained-release formulations,depots and the like. The composition can be formulated as a suppository,with traditional binders and carriers such as triglycerides. Oralformulations can include standard carriers such as pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate etc. Examples of suitable carriers aredescribed in “Remington's Pharmaceutical Sciences,” Martin. Suchcompositions will contain an effective amount of the immunogenpreferably in purified form, together with a suitable amount of carrierso as to provide the form for proper administration to the patient. Asknown in the art, the formulation will be constructed to suit the modeof administration.

Therapeutic formulations of the product may be prepared for storage aslyophilized formulations or aqueous solutions by mixing the producthaving the desired degree of purity with optional pharmaceuticallyacceptable carriers, diluents, excipients or stabilizers typicallyemployed in the art, i.e., buffering agents, stabilizing agents,preservatives, isotonitiers, non-ionic detergents, antioxidants andother miscellaneous additives, see Remington's Pharmaceutical Sciences,16th ed., Osol, ed. (1980). Such additives are generally nontoxic to therecipients at the dosages and concentrations employed, hence, theexcipients, diluents, carriers and so on are pharmaceuticallyacceptable.

An immune refocused polypeptide (which includes an antigen, a portionthereof, an epitope, a determinant and so on, which can be produced as asubunit substantially free of contaminating proteins, including otherinfluenza proteins, in combination with other viral or non-viralpolypeptides; as an IR polypeptide of interest which can be expressed orproduced in recombinant viruses, VLP's or in combination with one ormore proteins of virus or cell origin; as an IR polypeptide which can beexpressed or produced as an isolated molecule and then combined with oneor more proteins of virus or cell origin; and so on) can be obtained ormade in substantially pure form. An “isolated” or “purified” immunogenis substantially free of contaminating proteins from the medium fromwhich the immunogen is obtained, or substantially free of chemicalprecursors or other chemicals in the medium used which containscomponents that are chemically synthesized. The language “substantiallyfree of subcellular material” includes preparations of a cell in whichthe cell is disrupted to form components which can be separated fromsubcellular components of the cells, including dead cells, and portionsof cells, such as cell membranes, ghosts and the like, from which theimmunogen is isolated or recombinantly produced. Thus, an immunogen thatis substantially free of subcellular material includes preparations ofthe immunogen having less than about 30%, 25%, 20%, 15%, 10%, 5%, 2.5%or 1%, (by dry weight) of subcellular contaminants, or any other elementthat differs from the product of interest.

As used herein, the terms “stability” and “stable” in the context of aliquid formulation comprising an immunogen refer to the resistance ofthe immunogen in a formulation to thermal and chemical aggregation,degradation or fragmentation under given manufacture, preparation,transportation and storage conditions, such as, for one month, for twomonths, for three months, for four months, for five months, for sixmonths or more. The “stable” formulations of the invention retainbiological activity equal to or more than 80%, 85%, 90%, 95%, 98%, 99%/oor 99.5% under given manufacture, preparation, transportation andstorage conditions. The stability of said immunogen preparation can beassessed by degrees of aggregation, degradation or fragmentation bymethods known to those skilled in the art, including, but not limitedto, physical observation, such as, with a microscope, particle size andcount determination and so on, compared to a reference.

The term, “carrier,” refers to a diluent, adjuvant, excipient or vehiclewith which the therapeutic is administered. Such physiological carrierscan be sterile liquids, such as water and oils, including those ofpetroleum, animal, vegetable or synthetic origin, such as peanut oil,soybean oil, mineral oil, sesame oil and the like. Water is a suitablecarrier when the pharmaceutical composition is administeredintravenously. Saline solutions and aqueous dextrose and glycerolsolutions also can be employed as liquid carriers, particularly forinjectable solutions. Suitable pharmaceutical excipients include starch,glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silicagel, sodium stearate, glycerol monostearate, talc, sodium chloride,dried skim milk, glycerol, propylene glycol, ethanol and the like. Thecomposition, if desired, can also contain minor amounts of wetting oremulsifying agents, or pH buffering agents. The carrier can include asalt and/or buffer.

Buffering agents help to maintain the pH in the range which approximatesphysiological conditions. Buffers are preferably present at aconcentration ranging from about 2 mM to about 50 mM. Suitable bufferingagents for use with the instant invention include both organic andinorganic acids, and salts thereof, such as citrate buffers (e.g.,monosodium citrate-disodium citrate mixture, citric acid-trisodiumcitrate mixture, citric acid-monosodium citrate mixture etc.), succinatebuffers (e.g., succinic acid-monosodium succinate mixture, succinicacid-sodium hydroxide mixture, succinic acid-disodium succinate mixtureetc.), tartrate buffers (e.g., tartaric acid-sodium tartrate mixture,tartaric acid-potassium tartrate mixture, tartaric acid-sodium hydroxidemixture etc.), fumarate buffers (e.g., fumaric acid-monosodium fumaratemixture, fumaric acid-disodium fumarate mixture, monosodiumfumarate-disodium fumarate mixture etc.), gluconate buffers (e.g.,gluconic acid-sodium glyconate mixture, gluconic acid-sodium hydroxidemixture, gluconic acid-potassium gluconate mixture etc.), oxalatebuffers (e.g., oxalic acid-sodium oxalate mixture, oxalic acid-sodiumhydroxide mixture, oxalic acid-potassium oxalate mixture etc.), lactatebuffers (e.g., lactic acid-sodium lactate mixture, lactic acid-sodiumhydroxide mixture, lactic acid-potassium lactate mixture etc.) andacetate buffers (e.g., acetic acid-sodium acetate mixture, aceticacid-sodium hydroxide mixture etc.). Phosphate buffers, carbonatebuffers, histidine buffers, trimethylamine salts, such as Tris, HEPESand other such known buffers can be used.

Preservatives may be added to retard microbial growth, and may be addedin amounts ranging from 0.2%-1% (w/v). Suitable preservatives for usewith the present invention include phenol, benzyl alcohol, m-cresol,octadecyldimethylbenzyl ammonium chloride, benzyaconium halides (e.g.,chloride, bromide and iodide), hexamethonium chloride, alkyl parabens,such as, methyl or propyl paraben, catechol, resorcinol, cyclohexanoland 3-pentanol.

Isotonicifiers are present to ensure physiological isotonicity of liquidcompositions of the instant invention and include polhydric sugaralcohols, preferably trihydric or higher sugar alcohols, such asglycerin, erythritol, arabitol, xylitol, sorbitol and mannitol.Polyhydric alcohols can be present in an amount of between about 0.1% toabout 25%, by weight, preferably about 1% to about 5% taking intoaccount the relative amounts of the other ingredients.

Stabilizers refer to a broad category of excipients which can range infunction from a bulking agent to an additive which solubilizes thetherapeutic agent or helps to prevent denaturation or adherence to thecontainer wall. Typical stabilizers can be polyhydric sugar alcohols;amino acids, such as arginine, lysine, glycine, glutamine, asparagine,histidine, alanine, ornithine, L-leucine. 2-phenylalanine, glutamicacid, threonine etc.; organic sugars or sugar alcohols, such as lactose,trehalose, stachyose, arabitol, erythritol, mannitol, sorbitol, xylitol,ribitol, myoinisitol, galactitol, glycerol and the like, includingcyclitols such as inositol; polyethylene glycol; amino acid polymers;sulfur containing reducing agents, such as urea, glutathione, thiocticacid, sodium thioglycolate, thioglycerol, α-monothioglycerol and sodiumthiosulfate; low molecular weight polypeptides (i.e., <10 residues);proteins, such as human serum albumin, bovine serum albumin, gelatin orimmunoglobulins; hydrophilic polymers, such as polyvinylpyrrolidone,saccharides, monosaccharides, such as xylose, mannose, fructose orglucose; disaccharides, such as lactose, maltose and sucrose;trisaccharides, such as raffinose; polysaccharides, such as, dextran andso on. Stabilizers can be present in the range from 0.1 to 10,000 w/wper part of immunogen.

Additional miscellaneous excipients include bulking agents, (e.g.,starch), chelating agents (e.g., EDTA), antioxidants (e.g., ascorbicacid, methionine or vitamin E) and cosolvents.

As used herein, the term “surfactant” refers to organic substanceshaving amphipathic structures, namely, are composed of groups ofopposing solubility tendencies, typically an oil-soluble hydrocarbonchain and a water-soluble ionic group. Surfactants can be classified,depending on the charge of the surface-active moiety, into anionic,cationic and nonionic surfactants. Surfactants often are used aswetting, emulsifying, solubilizing and dispersing agents for variouspharmaceutical compositions and preparations of biological materials.

Non-ionic surfactants or detergents (also known as “wetting agents”) maybe added to help solubilize the therapeutic agent, as well as to protectthe therapeutic protein against agitation-induced aggregation, whichalso permits the formulation to be exposed to shear surface stresseswithout causing denaturation of the protein. Suitable non-ionicsurfactants include polysorbates (20, 80 etc.), polyoxamers (184, 188etc.), Pluronic® polyols and polyoxyethylene sorbitan monoethers(TWEEN-20®, TWEEN-80® etc.). Non-ionic surfactants may be present in arange of about 0.05 mg/ml to about 1.0 mg/ml, preferably about 0.07mg/ml to about 0.2 mg/ml.

As used herein, the term, “inorganic salt,” refers to any compound,containing no carbon, that results from replacement of part or all ofthe acid hydrogen or an acid by a metal or a group acting like a metal,and often is used as a tonicity adjusting compound in pharmaceuticalcompositions and preparations of biological materials. The most commoninorganic salts are NaCl, KCl, NaH₂PO₄ etc.

The present invention can provide liquid formulations of an immunogenhaving a pH ranging from about 5.0 to about 7.0, or about 5.5 to about6.5, or about 5.8 to about 6.2, or about 6.0, or about 6.0 to about 7.5,or about 6.5 to about 7.0.

The instant invention encompasses formulations, such as, liquidformulations having stability at temperatures found in a commercialrefrigerator and freezer found in the office of a physician orlaboratory, such as from about −20° C. to about 5° C., said stabilityassessed, for example, by microscopic analysis, for storage purposes,such as for about 60 days, for about 120 days, for about 180 days, forabout a year, for about 2 years or more. The liquid formulations of thepresent invention also exhibit stability, as assessed, for example, byparticle analysis, at room temperatures, for at least a few hours, suchas one hour, two hours or about three hours prior to use.

Examples of diluents include a phosphate buffered saline, buffer forbuffering against gastric acid in the bladder, such as citrate buffer(pH 7.4) containing sucrose, bicarbonate buffer (pH 7.4) alone, orbicarbonate buffer (pH 7.4) containing ascorbic acid, lactose, oraspartame. Examples of carriers include proteins, e.g., as found in skimmilk, sugars, e.g., sucrose, or polyvinylpyrrolidone. Typically thesecarriers would be used at a concentration of about 0.1-90% (w/v) butpreferably at a range of 1-10% (w/v).

The formulations to be used for in vivo administration must be sterile.That can be accomplished, for example, by filtration through sterilefiltration membranes. For example, the subcellular formulations of thepresent invention may be sterilized by filtration.

The immunogen composition will be formulated, dosed and administered ina manner consistent with good medical practice. Factors forconsideration include severity of the disease, the particular mammalbeing treated, the clinical condition of the individual patient, thecause of the disorder, the site of delivery of the agent, the method ofadministration, the scheduling of administration, and other factorsknown to medical practitioners. The “therapeutically effective amount”of the immunogen thereof to be administered will be governed by suchconsiderations, and can be the minimum amount necessary to prevent,ameliorate or treat a targeted disease, condition or disorder.

The amount of antigen is an amount sufficient to induce the desiredhumoral and/or cell mediated immune response in the target host. Theamount of immunogen of the present invention to be administered willvary depending on the species of the subject, physical characteristicsof the host, such as age, weight and so on, preferred mode of deliveryand so on. Generally, the dosage employed can be about 10 to about 1500μg/dose. In comparison, the current subunit preparations containelements from three subtypes of virus. The trivalent vaccines generallycontain about 7 to about 25 μg of HA from each of the three contributingstrain. That can serve as a starting point for titrating the vaccinecomposition of interest.

As used herein, the term “effective amount” refers to the amount of atherapy (e.g., a prophylactic or therapeutic agent), which is sufficientto reduce the severity and/or duration of a targeted disease, ameliorateone or more symptoms thereof, prevent the advancement of a targeteddisease or cause regression of a targeted disease, or which issufficient to result in the prevention of the development, recurrence,onset, or progression of a targeted disease or one or more symptomsthereof. For example, a treatment of interest can increase survivabilityof the host or reduce the severity of disease, based on baseline or anormal level, by at least 5%, preferably at least 10%, at least 15%, atleast 20%, at least 25%, at least 30%, at least 35%, at least 40%, atleast 45%, at least 50%, at least 55%, at least 60%, at least 65%, atleast 70%, at least 75%, at least 80%, at least 85%, at least 90%, atleast 95%, or at least 100%. In another embodiment, an effective amountof a therapeutic or a prophylactic agent reduces the symptoms of atargeted disease, such as a symptom of influenza or duration of illnessby at least 5%, preferably at least 10%, at least 15%, at least 20%, atleast 25%, at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 90%, at least 95%, or atleast 100%. Also used herein as an equivalent is the term,“therapeutically effective amount.”

Where necessary, the composition may also include a solubilizing agentand a local anesthetic such as lidocaine or other “caine” anesthetic toease pain at the site of the injection.

Generally, the ingredients are supplied either separately or mixedtogether in unit dosage form, for example, as a dry lyophilized powderor water-free concentrate in a sealed container, such as an ampule orsachet indicating the quantity of active agent. Where the composition isto be administered by infusion, it can be dispensed with an infusionbottle containing sterile pharmaceutical grade water or saline. Wherethe composition is administered by injection, an ampule of sterile waterfor injection or saline can be provided, for example, in a kit, so thatthe ingredients may be mixed prior to administration. Alternatively, theampoule can comprise a fluid containing the active agent of interest,for example, as a concentrate for dilution prior to use or in a formready for administration.

An article of manufacture containing materials useful for the treatmentof the disorder described above is provided. The article of manufacturecan comprise a container and a label. Suitable containers include, forexample, bottles, vials, syringes and test tubes. The containers may beformed from a variety of materials such as glass or plastic. Thecontainer holds a composition of interest and may have a sterile accessport (for example, the container may be an intravenous solution bag or avial having a stopper pierceable by a hypodermic injection needle). Thelabel on or associated with the container indicates that the compositionis used for treating influenza. The article of manufacture may furthercomprise a second container comprising a pharmaceutically acceptablebuffer, such as phosphate-buffered saline, Ringer's solution or dextrosesolution. It may further include other materials desirable from acommercial and user standpoint, including buffers, diluents, filters,needles, syringes and package inserts with instructions for use.

The instant invention also includes kits, e.g., comprising animmunogenic composition of interest, homolog, derivative thereof and soon, for use, for example, as a vaccine, and instructions for the use ofsame and so on. The instructions may include directions for preparingthe composition, derivative and so on. The composition can be in liquidform or presented as a solid form, generally, desiccated or lyophilized.The kit can contain suitable other reagents, such as a buffer, areconstituting solution and other necessary ingredients for the intendeduse. A packaged combination of reagents in predetermined amounts withinstructions for use thereof, such as for a therapeutic use iscontemplated. In addition, other additives may be included, such as,stabilizers, buffers and the like. The relative amounts of the variousreagents may be varied to provide for concentrates of a solution of areagent, which provides user flexibility, economy of space, economy ofreagents and so on. The kit can comprise a delivery means, such as adevice containing a needle, such as a syringe, which, optionally can bepreloaded with the composition of interest for delivery when needed.

Citation of any of the references discussed hereinabove shall not beconstrued as an admission that any such reference is prior art to thepresent invention. All references cited herein are herein incorporatedby reference in entirety.

The invention now will be exemplified by the following non-limitingexamples.

Example 1

Eight immune dampened and refocused hemagglutinin genes derived from theWyoming strain (H3N2) were designed and engineered as described above.For example, nucleotides were substituted by site-directed mutagenesisto introduce N-linked sequons leading to complex carbohydratemodifications, and/or deletions and/or charge changes of the amino acidsinto the five major immunogenic and highly variable sites containing theIDNPEs.

Introduction of N-linked sequons was used to maximize the size of theimmune dampening by each change, particularly in the larger antigenicsites while reducing the number of wild type amino acid changes requiredto dampen while minimizing any impact on the conformational complexityof the glycoprotein and receptor binding domain. In some cases, as fewas three amino acid changes were needed. Antigenic Site B (187-196)targets both the B cell and CD4 helper T cell IDNPEs.

To expedite the study, both DNA and protein subunit vaccines wereengineered. For DNA immunization, full-length hemagglutinin genes werecloned into the pTriEx vector (Invitrogen) behind the cytomegalovirus(CMV) promoter. Transient transfection of mammalian cells with thepTriEx-IHA constructs demonstrated that full-length hemagglutinin genesresembling native viral proteins were expressed as trimers and could besolublized from plasma membrane extracts.

Nine groups of outbred mice were immunized with the DNA constructscontaining the eight mutated and one unmodified full length wild type HAglycoproteins. A tenth group was immunized with the empty pTriEx vectorfor a negative control.

In addition to the DNA expression vectors, recombinant protein wasproduced for immunization. The HA ectodomain contains the domains forthe assembly of the trimeric glycoprotein spike and binding the hostcell receptor. In addition, removal of the membrane spanning andcytoplasmic domains causes recombinant HA trimers to be released intothe culture supernatant. Therefore, each of the mutated HA genes wastruncated at the end of the ectodomain and cloned into a vector havingthe phage T7 promoter. Transfection of the ectodomain vectors into cellsinfected with a recombinant vaccinia virus that expressed the phage T7RNA polymerase resulted in the production of HA trimers which weresecreted into the culture media. The ectodomain trimers were purifiedfor use as protein immunogens.

Mice were pre-bled. One group of mice was used as a negative control andthe other was immunized with unmodified (wild type) antigens.

In another set of experiments, mice in the principal groups wereimmunized by injection of 10 micrograms of DNA (in 0.1 mL sterile water)of mutated HA glycoproteins into each quadriceps muscle. After a rest of5 weeks, the mice were boosted with a second DNA immunization. Afteranother 4-5 weeks, the mice were again boosted by two subcutaneousimmunizations of 10 micrograms each of purified ectodomain glycoprotein.The first protein immunization was formulated in Complete Freund'sAdjuvant and the second in Incomplete Freund's Adjuvant. Two weeksfollowing the final immunization, the mice were euthanized and bled outfor serum.

The sera were tested for 1) reactivity to mutant and wild type HAproteins in Western blot and ELISA formats, 2) recognition of linearepitopes by peptide ELISA, 3) protection of conformational epitopes fromdegradation by proteases, and 4) functional testing by hemagglutinationinhibition and virus neutralization of homologous and heterologousinfluenza strains.

Sera from mice immunized with the panel of immune refocused HA subunitengineered antigens resulted in the generation of high titer antisera asmeasured by an HA-specific ELISA. All groups of mice exhibited titers towild type HA in the range of 1:100-300,000. Down selection of thevarious mutated HA glycoproteins were made based on the ability of theantisera to exhibit cross subtype HI antibody in a standard HI assay.

Mutants A2, B1, B2, B3, CE, CEB4, CEB5, and D1 of H3N2 A/Wyoming/03/2003gave equal to or higher cross subtype HI and/or virus neutralizationtiters against a panel of heterologous virus subtypes used in the assay.Thus, immune dampening and refocusing resulted in the production of HAglycoprotein subunit vaccine candidates capable of inducingsignificantly improved cross-subtype anti-viral protection as measuredin vitro by standardized and accepted surrogate HI and virusneutralization assays.

Mutant A2 is the mutation in the A epitope of HA wherein KRRSNKS (SEQ IDNO:1) is replaced by GG. BI is the mutation in the B epitope of HAwherein a glycosylation site is introduced at amino acid 197 (QIS toNAS). B2 is the mutation in the B epitope of HA wherein a glycosylationsite is introduced at amino acid 189 (SDQ to NVT). B3 is the mutation inthe B epitope of HA wherein a glycosylation site is introduced at aminoacid 193 (SLY to NIT). CE contains two mutations, a glycosylation siteis introduced into the C epitope at position 276 (KCN→NCT) and aglycosylation site is added into the E epitope at position 83 (NKK→NKT).CEB4 is CE with an additional mutation in the B epitope, a glycosylationsite is added at position 158 (KYK→NST). CEB5 is the CE with anadditional mutation in the B epitope, a glycosylation site was added atposition 159. D1 is the mutation in the D epitope of HA wherein aglycosylation site is introduced at amino acid 201 (RIT to NIT).

In another set of experiments, refocused polypeptide antigens weretested for hemagglutinin inhibition titer and serum neutralization titerwhen compared to different strains of H3N2 virus. The mutants werederived from the A/Wyoming/2003 strain. M3 has the B2 epitope; M5 hasthe CE epitopes; and M6 has the B4CE epitopes as described above. Micewere exposed to the various muteins, A/Wyoming/2003 strain virus as thewild type positive control and carrier alone as the negative control.Mouse serum was then tested for hemagglutinin inhibition titers againstthree strains, the cognate Wyoming strain, Panama/1999 andWellington/2004 strains. Other mouse serum was tested for serumneutralization titers against the cognate Wyoming strain, Korea/2003,Brisbane/9/2006 and Brisbane/10/2007 strains. Control sera from miceexposed to carrier alone generated no specific hemagglutinin inhibitionantibody that reacted with the Wyoming, Panama and Wellington strains(titer=10). Mice exposed to wild type Wyoming virus generated antiserumreactive with the Wyoming and Wellington strains (titer=1280), andmarginally with the Panama strain (titer=226). The M5 mutant producedantisera that reacted twice as vigorously as wild type with the Wyomingand Wellington strains (titer=2560) and just slightly less with thePanama strain (titer=1920). The M6 mutant generated antisera thatreacted at about the same level as did the M5 mutant with Panama andWyoming strains (titers=2560 and 1280, respectively). The M6 mutanthowever generated a high inhibiting antiserum with a titer four timeshigher than all other titers, when exposed to the Wellington strain(titer=10240). Thus, immunorefocusing resulted in broadened responsesagainst two other strains aside from the cognate strain, along with avery high response against the Wellington strain when the triplemodified mutein was used. In the neutralization studies, mice exposed tocarrier produced no specific antibody. Mice exposed to Wyoming generatedantisera that reacted strongly with Wyoming (titer=640); the titer forKorea and Brisbane 2006 was a quarter that of Wyoming (titer=160); andthere was essentially no reactivity with Brisbane 2007 (titer=20). TheM3 mutein generated in mice antisera that was four time as reactive aswild type immunogen on Wyoming, Brisbane 2006 and Brisbane 2007(titer=2560). That antisera did not react with Korea (titer=3). Miceexposed to M5 generated antisera reactive with Wyoming (titer=80), wastwice as reactive with Brisbane 2006 (titer=160) and thirty times asreactive with Korea (titer=2560). That antisera was substantiallyunreactive with Brisbane 2007 (titer=20). Thus, broadened responses tothree other strains were obtained with the immune refocused antigens ofinterest.

Example 2

The safety, toxicity and potency of recombinant immunogens are evaluatedaccording to the guidelines in 21 CFR 610, which include: (i) generalsafety tests, as well as acute and chronic toxicity tests.

Immunogenicity data are derived from an accepted animal model thatresponds well to human influenza vaccine (e.g. guinea pigs, mice,ferrets or cotton rats). The investigations include an evaluation ofimmune responses according to dose and dose intervals using vaccine thatcontains the strain intended for the final product. Immunogenicitystudies in relevant animal models are used to document consistency ofproduction, in particular during the validation phase of a vaccine fornovel influenza viruses manufacturing process. Suitable non-clinicalendpoints selected for the animal studies include death, weight loss,virus excretion rates, clinical signs such as fever, oculo-nasalsecretions and so on.

Groups of ferrets or other suitable animals are inoculatedintraperitoneally with 100 μl of immunogen containing 300 μg of theimmunogen of interest. Suitable negative and positive controls are used.

The animals are monitored for general health and body weight for 14 dayspost infection. Similar to animals that receive placebo, animals thatreceive the immunogen remain healthy, and do not lose weight or displayovert signs of disease during the observation period.

For the more stringent safety test, groups of animals are injected with300 μg of the immunogen.

One day after inoculation, 3 animals in each group are euthanized andthe spleen, lung and liver homogenates are analyzed for immunogen. Atweek 4, 8, 12, and 16 post-infection, 3 animals in each group areeuthanized and spleen, liver and lung homogenates are obtained andanalyzed to assess presence of the immunogen.

The immunogen is deemed safe if no adverse health effects are observedand the animals gain weight at the normal rate compared to animalsinoculated with placebo as an internal control.

To evaluate the acute and chronic toxicity of an immunogen, groups offerrets are inoculated intradermally with 300 μg of the immunogen atgraded doses or saline.

Three days post-inoculation, 8 animals in each group are euthanized toaccess the acute effects of the immunogen on the animals. At 28 dayspost-inoculation, the remaining 8 animals in each group are euthanizedto evaluate any chronic effects on the animals. At both time points, thebody weight of each animal is obtained. In addition, the gross pathologyand appearance of the injection sites are examined. Blood is taken forblood chemistry, and the histopathology of the internal organs andinjection sites are performed at each time point.

Other animals are given a total of 3 doses of vaccine at 0, 14 and 60days and the immune response to hemagglutinin is measured by ELISA usingsera collected from the animals at 10 day intervals. The neutralizationof influenza virus is measured in the collected sera, for example, 80days after the first vaccination.

It should be understood that various changes and modifications to thepresently preferred embodiments described herein will be apparent tothose skilled in the art. Such changes and modifications can be madewithout departing from the spirit and scope of the present subjectmatter and without diminishing its intended advantages. It is thereforeintended that such changes and modifications be embraced by the appendedclaims.

REFERENCES

-   Thomas Francis, Jr. in Proceedings of the American Philosophical    Society, Vol. 104, No. 6 (Dec. 15, 1960), pp. 572-578, according to    The Swine Flu Episode and the Fog of Epidemics by Richard Krause in    DCD's Emerging Infectious Diseases Journal Vol. 12, No. 1 Jan. 2006    published Dec. 20, 2005.-   Garrity, R. R., G. Rimmelzwaan, A. Minassian, W. P. Tsai, G.    Lin, J. J. de Jong, J. Goudsmit, and P. L. Nara. 1997. Refocusing    neutralizing antibody response by targeted dampening of an    immunodominant epitope. J. Immunol. 159:279-89.-   Kohler H, Goudsmit, J. Nara P. Clonal dominance: cause for a limited    and failing immune response to HIV-1 infection and vaccination. J.    Acquir. Immune Defic. Syndr. 1992; 5(11):1158-68.-   Andreansky, S. S., John Stambas, Paul G. Thomas, Weidong Xie,    Richard J. Webby, and Peter C. Doherty Consequences of    immunodominant epitope deletion for minor influenza virus-specific    CD8⁺ T cell responses. J. Virol. 2005 April; 79(7):4329-39.-   Nara, P. L., and R. Garrity. 1998. Deceptive imprinting: a    cosmopolitan strategy for complicating vaccination. Vaccine    16:1780-7.-   Nara, P. L., R. R. Garrity, and J. Goudsmit. 1991. Neutralization of    HIV-1: a paradox of humoral proportions. FASEB J. 5:2437-55.-   Nara, P. L., and G. Lin. 2005. HIV-1: the confounding variables of    virus neutralization. Curr. Drug Targets Infect. Disord. 5:157-70.-   Trujiollo, J. D., N. M. Kumpula-McWhirter, K. J. Hotzel, M.    Gonzalez, and W. P. Cheevers. 2004. Glycosylation of immunodominant    linear epitopes in the carboxy-terminal region of the caprine    arthritis-encephalitis virus surface envelope enhances    vaccine-induced type-specific and cross-reactive neutralizing    antibody responses. J. Virol. 78:9190-202.

The invention is claimed as follows:
 1. An isolated compositioncomprising: (a) a cross reactive epitope that stimulates an immuneresponse to influenza virus; and (b) an immunodominant surface epitope,that is immunodampened, of influenza virus hemagglutinin, wherein (i)said dampened immunodominant surface epitope is not as immunodominant assaid immunodominant surface epitope in a wild type influenza virus; and(ii) said cross reactive epitope is subdominant in a wild type influenzavirus.
 2. The composition of claim 1 wherein said surface epitopecomprises hemagglutinin epitopes A-E.
 3. The composition of claim 1,further comprising a neuraminidase.
 4. The composition of claim 1comprising an influenza virus particle.
 5. The composition of claim 4,wherein said particle is inactivated.
 6. The composition of claim 1wherein said immunodampened surface epitope comprises addition orremoval of a glycosylation site.
 7. The composition of claim 1, whereinsaid immunodampened surface epitope comprises an amino acid addition,substitution or deletion.
 8. The composition of claim 1 comprising avirus-like particle.
 9. The composition of claim 1 expressed on thesurface of influenza virus.
 10. The composition of claim 1, furthercomprising a pharmaceutically acceptable carrier, diluent or excipient.11. The composition of claim 1, wherein said immunodampened epitopecomprises one or more mutations.
 12. The composition of claim 1, whereinsaid immunodampened epitope comprises one or more mutations selectedfrom the group consisting of an amino acid addition, an amino acidsubstitution, and an an amino acid deletion.
 13. The composition ofclaim 1, wherein said influenza virus comprises influenza A, influenza Bor influenza C.
 14. A recombinant virus comprising one or more of thecomposition of claim
 1. 15. A virus comprising one or more of thecomposition of claim
 1. 16. An immunogenic composition comprising one ormore of the composition of claim
 1. 17. The composition of claim 1,further comprising an adjuvant.
 18. The composition of claim 1, whereinsaid influenza virus is H3N2.
 19. The composition of claim 2 whereinsaid immunodampened epitope comprises: a. NVT replacing SDQ in epitopeB; b. NAS replacing QIS in epitope B; c. NCT replacing KCN in epitope C;d. NKT replacing NKK in epitope E; e. NST replacing KYK in epitope B; f.an additional glycosylation site at position 159 in epitope B; g. NITreplacing MT in epitope D; h. KRRSNKS (SEQ NO:1) of epitope A replacedby GG; i. SLY in epitope B replaced by NIT; j. YKY in epitope B replacedby NTS; k. a deletion of amino acids 201-220 of epitope D; l. replacingKRR in epitope A with AGA; m. replacing KYKY (SEQ ID NO:3) with AYKY(SEQ ID NO:35) in epitope B; n. replacing SDQI (SEQ ID NO:2) with SAQI(SEQ ID NO:36) in epitope B; o. replacing KCN with ACN in epitope C; p.replacing RIT with AIT in epitope D; q. replacing SLY with SLT inepitope B; r. replacing LYIW (SEQ ID NO:37) with VYIW (SEQ ID NO:38) inepitope ID4; s. replacing LYIW (SEQ ID NO:37) with VTIW (SEQ ID NO:39)in epitope ID4; t. replacing WEEP (SEQ ID NO:37) with IHAG (SEQ II)NO:40) in epitope ID4; u. replacing QNK in epitope E with NET; or v.combination thereof.